Hepatocyte growth factor (HGF), also known as scatter factor (SF), is a growth factor which is involved in various physiological processes, such as wound healing and angiogenesis. The high affinity interaction of HGF interaction with its receptor (c-Met) is implicated in tumour growth, invasion and metastasis.
c-Met has been shown to be involved in tumour growth, invasion and metastasis in many human cancers of epithelial origin. c-Met is expressed by most carcinomas and its elevated expression relative to normal tissue has been detected in cancers of: lung, breast, colorectal, pancreatic, head and neck, gastric, hepatocellular, ovarian, renal, glioma, melanoma and a number of sarcomas. In colorectal carcinoma (CRC), over-expression of c-Met has been detected in dysplastic aberrant crypt foci, the earliest pre-neoplastic lesions of the disease. In head and neck squamous cell cancer, c-Met is reportedly expressed or overexpressed in roughly 80% of primary tumours. In prostate cancer metastasis to bone, c-Met was reported overexpressed in over 80% of bone metastasis.
Under normal conditions, c-Met is expressed on epithelial cells and activated in a paracrine fashion, by mesenchymally derived HGF. The activation of c-Met in normal cells is a transient event and is tightly regulated. In tumour cells, however, c-Met can be constitutively active. In cancer, aberrant c-Met stimulation can be achieved through c-Met amplification/over-expression, activating c-Met mutations (e.g. structural alterations) and acquisition of autonomous growth control through creation of autocrine signalling loops. In addition, a defective down-regulation of the c-Met receptor will also contribute to aberrant c-Met expression in the cell membrane. While the over-expression of c-Met is HGF dependent (autocrine/paracrine), structural alterations caused by mutations are HGF independent (e.g. loss of extracellular domain).
Poethko et al [J. Nucl. Med., 45(5), 892-902 (2004)] disclose a method of radiolabelling peptides with the radioisotope 18F, wherein an aminoxy-functionalised peptide is condensed with [18F]-fluorobenzaldehyde to give a labelled peptide having an oxime ether linkage as follows:

Schottelius et al [Bioconj. Chem., 19(6), 1256-1268 (2008)] further developed the method of Poethko et al. Schottelius et al use an aminoxy-functionalised peptide wherein the amine of the aminoxy group is protected with an N-Boc (Boc=tert-butyloxycarbonyl) protecting group. The desired aminoxy-functionalised peptide is generated in situ in the presence of [18F]-fluorobenzaldehyde via deprotection of the N-Boc group at acidic pH (pH=2) at 75° C. Schottelius et al used a 5-fold molar excess of the Boc-protected precursor, because the deprotection was not quantitative under the reaction conditions.
Mezo et al [J. Pept. Sci., 17, 39-46 (2010)] describe some of the problems associated with the above oxime ligation chemistry of Boc-protected aminoxy-functionalised peptides. Thus, it is known that the Boc-aminoxy reagent can acylate formed Boc-protected aminoxy-peptide, leading to undesirable by-products. It is also known that the reactivity of the free aminoxy group of the functionalised peptide is high towards carbonyl compounds. Consequently, unwanted condensation can occur with any adventitious aldehydes or ketones present either in the reaction mixture or in any subsequent purification steps. Such aldehydes or ketones could be traces of acetone present in the solvents used, or formaldehyde (e.g. from plasticizers). Mezo et al are interested in solving this problem for both the conjugation of anti-cancer drugs and of [18F]-fluorobenzaldehyde to peptides. Mezo et al solve the problem by carrying out the deprotection of the Boc-aminoxy peptide in the presence of a tenfold molar excess of free (aminoxy)acetic acid (Aoa) as a ‘carbonyl capture agent’. The deprotected aminoxy-peptide and excess Aoa is then lyophilised and stored at 4° C. Immediately prior to the oxime ligation reaction, the lyophilised mixture is reconstituted, and excess Aoa is separated by HPLC or Sep-Pak plus C18 cartridge. Mezo et al provide an example in which non-radioactive (i.e. 19F) 4-fluorobenzaldehyde is conjugated to an aminoxy-functionalised somatostatin peptide using this technique. Mezo et al do not provide any data on 18F-radiolabelling.
WO 2012/022676 discloses an imaging agent which comprises an 18F-radiolabelled 18 to 30-mer c-Met binding cyclic peptide (cMBP) of Formula I:Z1-[cMBP]-Z2  (I)
where:                cMBP is of Formula II:-(A)x-Q-(A′)y-  (II)        where Q is the amino acid sequence (SEQ-1):        -Cysa-X1-Cysc-X2-Gly-Pro-Pro-X3-Phe-Glu-Cysd-Trp-Cysb-Tyr-X4-X5-X6-        wherein X1 is Asn, His or Tyr;                    X2 is Gly, Ser, Thr or Asn;            X3 is Thr or Arg;            X4 is Ala, Asp, Glu, Gly or Ser;            X5 is Ser or Thr;            X6 is Asp or Glu;            and Cysa-d are each cysteine residues such that residues a and b as well as c and d are cyclised to form two separate disulfide bonds;            A and A′ are independently any amino acid other than Cys, with the proviso that at least one of A and A′ is present and is Lys;                        x and y are independently integers of value 0 to 13, and are chosen such that [x+y]=1 to 13;        Z1 is attached to the N-terminus of cMBP, and is H or MIG;        Z2 is attached to the C-terminus of cMBP and is OH, OBc, or MIG,                    where Bc is a biocompatible cation;            each MIG is independently a metabolism inhibiting group which is a biocompatible group which inhibits or suppresses in vivo metabolism of the cMBP peptide;                        wherein cMBP is labelled at the Lys residue of the A or A′ groups with 18F.        
WO 2012/022676 also discloses that the imaging agents may be used as pharmaceutical compositions, wherein said compositions preferably comprises one or more radioprotectants preferably chosen from: ethanol; ascorbic acid; para-aminobenzoic acid (i.e. 4-aminobenzoic acid or pABA); gentisic acid (i.e. 2,5-dihydroxybenzoic acid), and salts of such acids with a biocompatible cation.
WO 2012/072736 discloses the use of alternative protecting group chemistry for the aminoxy groups of functionalised biomolecules. The protected aminoxy group is of formula:

wherein:
R1 and R2 are independently chosen from C1-3 alkyl, C1-3 fluoroalkyl or C4-6 aryl.
US 2013/0209358 A1 discloses that 18F-fluciclatide can undergo radiolysis at high radioactive concentration:

US 2013/0209358 A1 reports that 18F-fluciclatide is not stabilised by ethanol, and that ascorbic acid is not ideal for automated radiosyntheses, but that 4-aminobenzoic acid (or salt thereof) is effective. US 2013/0209358 A1 teaches that the 18F-fluciclatide is first prepared, and then the radioprotectant is added.
There is therefore still a need for improved methods of preparing and purifying 18F-labelled c-Met peptide radiotracers, to give high purity compositions suitable for radiopharmaceutical applications in vivo.